The present invention relates to novel long-acting prodrugs capable of undergoing chemical transformation in the body from an inactive into a bioactive form, said prodrugs bearing functional groups sensitive to mild basic conditions, more particularly fluorenylmethoxycarbonyl (Fmoc)- and fluorenylmethyl (Fm)-substituted prodrugs, and to pharmaceutical compositions comprising them.
Therapeutical drugs currently being used both in human therapy and veterinary can be classified according to various criteria. For example, drugs may be categorized as molecules having a proteinaceous-peptidic, i.e. composed of amino acid building units, or non-peptidic character, or by a criterion unrelated to the structure such as drugs absorbed orally or administered by other modes, i.e. injection, intranasal or topical, in order to reach the blood circulation.
Orally absorbed compounds are, in general, low molecular weight, rather stable, lipophilic (xe2x80x9coilyxe2x80x9d) and of a non-peptidic nature. Virtually all peptidic and protein drugs, due to their intrinsic hydrophilic (non-lipophilic) and polar features and metabolical instability, do not obey these criteria and must be administered mostly by injection. Further, as these molecules are rapidly degraded in the body by diverse mechanisms, notably proteolysis, they are usually short-living species.
Non-peptidic drugs are very often sufficiently hydrophobic and can reach the blood circulation through the gastrointestinal pathway. Due to their relative chemical stability, the non-peptidic drugs are usually long-living species.
Protein and peptide drugs have major and numerous clinical applications. e.g. insulin in the treatment of diabetes, gonadotropin-releasing hormone (GnRH) analogs in the therapy of prostate cancer, calcitonin in the treatment of bone-related disorders. The potential for this most important family of molecules is vast, but has as yet been only partially, if not marginally, explored. This, to large extent, is due to their short life in the body and inconvenience of the mode of administration. Non-peptidic drugs, such as antibiotics, although relatively long-lived, have to be administered several times a day over a period of a week, or longer, to maintain the desired continuous circulating levels.
Oral absorption of drugs is a highly desirable goal in the treatment of human diseases, particularly in prolonged therapeutical treatments. Structural alteration of drugs may result in the augmentation of oral and topical absorption, biostability and, eventually, bioavailability. Major efforts are currently being directed toward these goals. Most approaches include drug modification in such way that its native architecture, i.e. bioactive structure, is preserved. This native structure is the one recognized specifically by the drug""s target and is a prerequisite feature of the drug""s potency. Unfortunately, however, in many cases, the native structure is also recognized by the xe2x80x98clearing machinery systemxe2x80x99, which is capable of binding the drug, degrading or metabolizing it and thus accelerates its disposal. Thus, stabilization of the bioactive structure is often attempted concomitantly with the desire to enhance metabolic stability. Methods such as encapsulation, decreased solubility and chemical modification have been employed to achieve this goal.
It would be highly desirable to prolong the half-life of virtually many, if not all peptidic as well as non-peptidic drugs existing on the market or to be developed in the future, including antibiotics, antiviral, antihypertensive, antiinflammatory, analgesic, anticholesterolemia, anticarcinogenic, antidiabetic, growth-promoting, and other drugs. Prodrugs related to native drugs that are toxic above certain threshold concentrations might be particularly beneficial.
It is therefore an object of the present invention to provide novel prodrugs characterized by their high sensitivity to mild basic conditions and their capability of undergoing transformation from an inactive into a bioactive form under physiological conditions in the body.
It is another object of the present invention to provide prodrugs derived from drugs having free amino, carboxyl, hydroxyl and/or mercapto groups, said prodrugs being essentially non-active biologically but being capable of spontaneous and slow conversion to the original active drug molecule in the body, following administration.
It is still another object of the present invention to provide prodrugs that present higher metabolic stability and augmented bioavailability.
It is a further object of the present invention to provide prodrugs that represent alternative possibilities for drug administration, e.g. oral and transdermal, and are further capable of penetrating through physiological barriers, e.g. blood-brain-barrier.
It is still a further object of the present invention to provide prodrugs that permit specific drug targetting to inflicted locations in the body.
The present invention thus relates to novel prodrugs derivatized from a drug in which one or more groups of said drug molecule selected from the group comprising free amino, carboxyl, hydroxyl and/or mercapto, are substituted by functional groups sensitive to bases and removable under mild basic. e.g. physiological, conditions.
The new concept of the invention for slow-releasing drugs includes their derivatization into novel, generally more hydrophobic, drug derivatives. In this approach it is preferred to lose, rather than to preserve, the native conformation, the biological potency and the recognition identity of the drug by the degradative systems. An advantage of this approach, however, resides in the fact that the thus modified derivative can slowly and spontaneously hydrolyze back to the native active drug under the in vivo conditions.
In a particular embodiment, the prodrugs of the invention are of the formula:
Xxe2x80x94Y
wherein
Y is a moiety of a drug bearing at least one functional group selected from free amino, carboxyl, hydroxyl and/or mercapto, and
X is a radical selected from radicals of the formulas (i) to (iv): 
wherein R1 and R2, the same or different, are each hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, sulfo, amino, ammonium, carboxyl, PO3H2, or OPO3H2; R3 and R4, the same or different, are each hydrogen, alkyl or aryl; and A is a covalent bond when the radical is linked to a carboxyl or mercapto group of the drug Y, or A is OCOxe2x80x94 when the radical is linked to an amino or hydroxyl group of Y.
According to the present invention, Y is a moiety of any drug for human and veterinary use bearing at least one functional group selected from free amino, carboxyl, hydroxyl and/or mercapto, such as, but not being limited to, antidiabetic drugs, e.g. insulin; growth promoters, e.g. human growth hormone, bovine growth hormone; antibiotics such as aminoglycosides, e.g. gentamicin, neomycin and streptomycin, xcex2-lactams, such as penicillins, e.g. amoxicillin, ampicillin, piperacillin, and cephalosporins, e.g. cefaclor, cefminox and cephalexin, macrolides, e.g. carbomycin and erythromycin, and polypeptidic antibiotics. e.g. bacitracin, gramicidins and polymyxins; synthetic antibacterials, e.g. trimethoprim, piromidic acid, and sulfamethazine; analgesic and anti-inflammatory drugs. e.g. acetaminophen, aspirin, ibufenac, indomethacin; antiallergic and antiasthmatic drugs, e.g. amlexanox and cromolyn; antihypercholesterolemic drugs, e.g. clofibric acid, oxiniacic acid and triparanol; xcex2-adrenergic blockers and antihypertensive drugs, e.g. bupranolol, captopril, indenolol, propranolol and 4-aminobutanoic acid; antineoplastic drugs, e.g. daunorubicin, azacitidine, 6-mercaptopurine, interferons, interleukin-2, methotrexate, taxol and vinblastine; antiviral drugs, e.g. acyclovir, ganciclovir, amantadine, interferons, AZT and ribavirin, etc. The term drug according to the invention is intended to encompass also pheromones.
The terms xe2x80x9calkylxe2x80x9d, xe2x80x9calkoxyxe2x80x9d, xe2x80x9calkoxyalkylxe2x80x9d, xe2x80x9carylxe2x80x9d, xe2x80x9calkarylxe2x80x9d and xe2x80x9caralkylxe2x80x9d in the definitions of R1, R2, R3 and R4 herein are used to denote alkyl radicals of 1-8, preferably 1-4 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals of 6-10 carbon atoms, e.g. phenyl and naphthyl. The term xe2x80x9chalogenxe2x80x9d includes bromo, fluoro, chloro and iodo.
In a preferred embodiment of the invention, the functional group is the radical (i), wherein R1, R2, R3 and R4 are hydrogen and A is OCOxe2x80x94, i.e. the well-known 9-fluorenylmethoxycarbonyl (Fmoc) radical widely used in peptide synthesis for the temporary reversible protection of amino groups (for review, see L. A. Carpino, Acc. Chem. Res. (1987) 20,401-407). The Fmoc group is particularly suitable for peptide synthesis due to favorable synthetic manipulation for its introduction and removal, and preferential stability as a prerequisite for peptide synthesis and convenient purification. Furthermore, the related 9-fluorenylmethyl (Fm) group is also applicable for reversible masking of carboxylic functions, e.g. of amino acids. The resulting 9-fluorenylmethyl esters (Fm-esters) generate the parent free carboxylic functions following a xcex2-elimination reaction pathway upon mild basic treatment, and thus can be similarly employed for reversible masking of carboxylic functions of drugs. The Fmoc-group is of further potential similar use in the reversible protection of hydroxyl groups of tyrosine, serine and threonine.
The halogenated Fmoc radicals (i) wherein at least one of R1 and R2 is halogen in the 2 or 7 position, preferably Cl or Br, the 2-chloro-1-indenylmethoxycarbonyl (CLIMOC) radical (ii), the 1-benzo[f]indenylmethoxycarbonyl urethane (BIMOC) radical (iii), the urethane sulfone radical (iv) and corresponding radicals (i) to (iv) wherein A is a covalent bond, can be used similarly to Fmoc and Fm for substitution of free amino, carboxyl, hydroxyl and mercapto functions of drugs, thus providing a wide range of sensitivity toward removal of such groups under basic, e.g. physiological, conditions. In fact, the above radicals (i) to (iv) belong to a general family of rare chemical entities that undergo hydrolysis at neutral or slightly alkaline pH and mild conditions, and can therefore be used for temporary reversible protection of xcex1- and xcex5-amino groups, for example in peptide synthesis, and can be removed from the amino function by a xcex2-elimination reaction, under mild basic conditions.
According to the invention the radical (i) to (iv), preferably Fmoc covalently linked to amino and/or hydroxyl moieties, or Fm covalently linked to carboxyl and/or mercapto moieties, undergo hydrolysis (via xcex2-elimination) back to the free amino, hydroxy, mercapto or carboxyl functions, under physiological conditions in the body fluid, namely at pH 7.4 and 37xc2x0 C.
The prodrugs of the invention may be prepared by reaction of the drug molecule with a suitable reagent comprising a radical (i) to (iv). Several derivatives of 9-fluorenylmethyl (Fm) are available, such as 9-fluorenylmethyl-N-hydroxysuccinimide (Fmoc-OSu), a very specific reagent for amino functions; 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) that reacts with, and covalently attaches to, amino and hydroxyl radicals; 9-chloromethylfluorene (Fm-Cl) that reacts with mercapto radicals to yield S-Fm derivatives (Bodanszky and Bednarek, 1982); and 9-fluorenylmethanol (Fm-OH) that reacts with, and esterifies, carboxylic functions.
Functional groups sensitive to basic conditions which can be removed from amino, carboxylic, hydroxylic or mercapto functions following pathways different from xcex2-elimination are also available, but they usually require extensive manipulations not adequate for drug protection. One known exception is the trifluoroacetyl group (TFA) which is rather similar to Fmoc in its easy removal from amino groups, but it is potentially toxic, and thus TFA-dervatized drugs are not recommended for therapeutical purposes. Fmoc-aminoacids, e.g. Fmoc-leucine, on the other hand, were shown to have low index of toxicity in experimental animal models (Burch et al., 1991).
The invention further relates to pharmaceutical compositions comprising a prodrug according to the invention and a pharmaceutically acceptable carrier.